Modeling alcohol's effects on organs in animal models.

Researchers have developed numerous animal models to investigate the development of various alcohol-related diseases. Such models have provided insights into the mechanism through which alcohol can induce liver damage. Animal models also have helped researchers explore the mechanisms by which both short-term (e.g., binge) and long-term drinking can interfere with the function of the heart, a condition referred to as alcoholic cardiomyopathy. Furthermore, animal models have provided substantial information on the causes of fetal alcohol syndrome. Such models have demonstrated that exposure to alcohol during gestation can lead to prenatal and postnatal growth retardation, characteristic facial malformations, immune system deficiencies, and alterations in the central nervous system.

L ong-term (i.e., chronic) alcohol use affects almost eve ry organ system of the body, potentially resulting in serious illnesses, including l i ver disease, impaired heart function (i.e., card i o m yo p a t h y 1 ), and inflammation of the pancreas (i.e., pancre a t i t i s ) . Even one-time (i.e., acute) alcohol consumption, such as binge drinking, can temporarily alter the activity of many organ systems. Fu rt h e r m o re, heavy alcoh o l consumption by a pregnant woman can harm her fetus and lead to fetal abnormalities ranging from mild learning deficits to full-blown fetal alcohol synd rome (FAS). In vestigating the mechanisms underlying these adverse effects of alcohol consumption in humans f requently is impractical, because alcoholrelated disease generally develops only after many years of heavy drinking. Ot h e r studies would be unethical to conduct in humans. There f o re, re s e a rchers have been forced to use various animal models to gain insight into the processes re s p o n-s i b l e for alcohol's effects on the body and to determine new ways of pre ve n ting or treating alcohol-related diseases.
This article re v i ews numerous animal models used to explore alcohol's effects on several organ systems. First, it p rovides a brief ove rv i ew of the animal species and modes of alcohol administration used in such studies. The, art icle then summarizes the results of studies aimed at identifying the mechanisms underlying alcohol-induced liver damage, alcoholic card i o m yo p a t h y, and FA S .

Animal Species
To model alcohol's effects on the human b o d y, re s e a rchers ideally wish to use animals closely related to humans, such as nonhuman primates. Howe ve r, practical and economic considerations generally preclude the use of these animals. In one instance in which primates we re used as a model system, howe ve r, re s e a rchers studied baboons that consumed alcohol with their diets for several years. On e -t h i rd of these animals

BI D D A N D A C. PO N N A P PA, PH. D . , is an associate professor and EM A N U E L RU B I N, M . D . , is a professor and chairman in the De p a rtment of Pa t h o l o gy, Anatomy and Cell Bi o l o gy, Thomas Jefferson Un i ve r s i t y, Philadelphia, Pe n n s y l va n i a .
The pre p a ration of this article was supp o rted in part by National Institute on Alcohol Abuse and Alcoholism gra n t s AA-10967 and AA-07186. e ventually developed cirrhosis of the l i ver (Rubin and Lieber 1974), conclus i vely demonstrating alcohol's liver toxicity independent of nutritional factors. To date, howe ve r, this experiment re p res e n t s the only instance in which experimental alcohol administration induced cirrhosis in an animal model. Fu rt h e rm o re, the experiment lasted seve r a l years and led to disease development in only a minority of the animals studied. C o n s e q u e n t l y, the utility of this model for studying the pathogenesis of alcoholrelated diseases is limited.
Re s e a rchers have used numero u s other animals to test alcohol's effects, most commonly mammals, such as rats, mice, rabbits, hamsters, ferrets, and dogs. Some behavioral and genetic studies h a ve also been conducted in the fru i t fly D rosophila melanogaster and in the roundworm Ca e n o rhabditis e l e g a n s. Although the choice of the species depends on the nature of the experiment, most studies have been conducted in rats, owing to their manageable size , ease of handling, low cost, and the a vailability of extensive scientific data.

In Vi t ro Ap p ro a c h e s .
Many studies h a ve been conducted in vitro-that is, not with intact animals but with isolated organs, tissues, or cells. For example, re s e a rchers have used isolated, perf u s e d organs; tissue slices; suspensions of individual cells; cultures of cells new l y isolated from an organism (i.e., primary cell cultures); cells that have gained the ability to grow indefinitely (i.e., transformed cell lines); and isolated cell s t ru c t u res (i.e., organelles).
To study alcohol's effects on an organ (e.g., the liver or heart), re s e a rc h e r s sometimes isolate the particular organ and keep it functional by perfusing itthat is, by passing a fluid or medium t h rough it. Under these conditions, all the different cell types within that organ continue to function in their normal relationships with each other. This is p a rticularly important in organs that comprise several cell types, such as the l i ve r, which contains hepatocytes, endothelial cells, Kupffer cells, stellate cells, and bile duct epithelium, each of which has a specialized role in keeping the liver functioning. By perfusing such organs with fluids containing va r i o u s alcohol concentrations, re s e a rchers can study the coordinated response of all cell types to alcohol. At the same time, the influence of additional factors that might affect organ function in the intact organism (e.g., hormones) is eliminated in the perfused organ. A potential limitation of this approach is that such perfusion experiments last only a few hours b e f o re the organ fails.
In cell culture systems, cells derive d f rom mammalian organs (e.g., the live r or brain) or transformed cell lines (e.g., cells derived from certain tumors) are exposed to alcohol to study the cells' responses to this agent. In such systems, i n vestigators can easily manipulate the c e l l s' external environment (e.g., the c u l t u re medium or the temperature ) , t h e re by facilitating analyses of the interactions between alcohol and other molecules, such as hormones, neurotransmitters, and cytokines. In addition, the cultured cells are free from other influences, such as blood flow or the effects of neighboring cells of the same cell type or of another cell type in the same organ. This high degree of contro l over the environment allows re s e a rc h e r s to measure the intracellular re s p o n s e s of individual cells to perturbing agents, such as alcohol. Such analyses fre q u e n t l y i n vo l ve staining the cells with suitable d yes and then viewing the cells under p owe rful microscopes.

Modes of Alcohol Administration in Animal Models
The most physiological methods used to study alcohol's effects invo l ve acute or chronic alcohol administration to intact animals. Acute alcohol administration is a straightforw a rd pro c e d u re during which alcohol is injected into the abdomen (i.e., intraperitoneally) or delive red via a feeding tube dire c t l y into the stomach (i.e., intragastrically). In some studies, re s e a rchers have also injected alcohol into the ve i n s . C h ronic alcohol exposure of animals poses a greater challenge (for a re v i ew of the chronology of the development of various animal models in alcohol re s e a rc h , see Simpson and Peters 1993). In general, chronically exposed animals re c e i ve alcohol either with their diets (i.e., orally), i n t r a g a s t r i c a l l y, or by inhalation.

The Liquid Diet Mo d e l .
The simplest method for providing alcohol chro n ically is through the drinking water. Howe ve r, factors such as the animals' a version to alcohol, dehydration re s u l ting from insufficient fluid intake, nutritional imbalance, and low blood alcohol concentrations (BACs) re s u l ting from low consumption make this method unsuitable for many types of studies. To address these issues, De C a r l i and Lieber (1967) developed a liquid diet that was offered to rats as the only s o u rce of food and water (also see Lieber et al. 1989). In this diet, alcohol constitutes 36 percent of the total calories, with protein, carbohydrate, and fat accounting for 18, 11, and 35 perc e n t of total calories, re s p e c t i ve l y. Although rats generally dislike alcohol, when give n a choice of consuming the alcoholcontaining diet or starving, they choose the former and consume 14-18 grams of alcohol per kilogram bodywe i g h t ( g / k g ) per day, usually over a period of 4-6 weeks.
This treatment regimen induces the earliest form of liver damage (i.e., fatty l i ve r, which is characterized by fat deposits t h roughout the liver) and damage to a key type of liver cells (i.e., the hepatocytes), which can be detected with an e l e c t ron microscope. The tre a t m e n t does not, howe ve r, lead to the more serious forms of liver damage observe d in humans, such as death of liver tissue (i.e., necrosis), inflammation, or scarring of the liver (i.e., fibrosis) (De C a r l i and Lieber 1967). Howe ve r, baboons fed a modified Lieber-DeCarli liquid diet for several years did develop cirrhos i s ( Rubin and Lieber 1974). Since its d e velopment, the Lieber-DeCarli model has been widely used for studies of alcoh o l's effects on a variety of other organs, including the heart, brain, and pancre a s .

The In t ragastric Mo d e l .
Ts u k a m o t o and cow o rkers (1986) developed the intragastric model for inducing live r damage in rats, based on the hypothesis that rats have a higher rate of alcohol b re a k d own (i.e., metabolism) than do humans and may re q u i re sustained higher BACs than do humans to induce l i ver damage. In this model, alcohol in a liquid diet (i.e., the Ts k u k a m o t o -French diet) is infused directly into the stomach for several months using a surgically implanted catheter. This pro c ed u re achieves BACs of 50-80 millimolar (mM) (230-370 mg/dL) and thus e xceeds the BACs of 20-40 mM (90-180 mg/dL) obtained with the Lieber-DeCarli model. Rats treated with alcohol using this method develop fatty live r, l o c a l i zed necrosis, and mild portal fibros i s (i.e., fibrosis in the vicinity of small bile ducts through which the live r s e c retes bile into the gallbladder and small intestine) (Tsukamoto et al. 1990), but do not d e velop cirrhosis or other i r re versible changes.
The model's ability to reflect alcoh o l's effect on the human live r, howe ve r, is uncertain. For example, one mechanism through which alcohol induces liver damage is by incre a s i n g ox i d a t i ve stress in the cells-that is, by p roducing increased amounts of highly re a c t i ve, oxygen-containing molecules (i.e., oxygen radicals) or by re d u c i n g the levels of antioxidants that scave n g e those radicals. (For more information on ox i d a t i ve stress, oxygen radicals, and a n t i oxidants, see the sidebar, p. 96.) The Ts u k a m o t o -French diet has a high content of unsaturated fats and iron as well as a low content of carbohyd r a t e s , a combination that generates substantial ox i d a t i ve stress, there by exacerbating the alcohol-induced ox i d a t i ve s t ress. Although such considerations point to potential problems in extrapolating accurately from experimental data obtained in animal models to human alcoholic liver disease, such models do provide important information re g a rding the mechanisms underlying alcohol's damaging effects at the cellular leve l .
Mo re re c e n t l y, Enomoto and colleagues (1999) re p o rted a new rat model in which female Wistar rats re c e i ved 5 g/kg alcohol intragastrically eve ry 24 hours. After 4 weeks, this tre a t m e n t induced fat accumulation (i.e., steatosis), inflammation, and necrosis in the live r. Other re s e a rchers, howe ve r, still must re p roduce and confirm this model to establish its va l i d i t y.

The Inhalation Mo d e l .
The administration of alcohol vapor has been used mostly to study behavioral changes assoc i a t e d with alcohol withdrawal (Go l d s t e i n and Pal 1971); howe ve r, it has not been widely accepted for inducing organ damage. One drawback of the inhalation model is that it generates a nutritional imbalance between alcohol-exposed and control animals, because intox i c a t e d animals consume less food than do c o n t rol animals.

Animal Models of Alcohol-Induced Liver Damage
The live r, which plays a vital role in maintaining the internal enviro n m e n t (i.e., the homeostatic balance) in higher organisms, is one of the primary organs affected by alcohol abuse. Alcohol affects the liver both acutely and after long-term alcohol exposure. In humans, depending on the extent of alcohol abuse, alcoholic l i ver disease pro g resses through va r i o u s stages, from fatty liver to alcoholic hepatitis and finally to cirrhosis. Howe ve r, the fact that only 15-20 percent of heavy drinkers develop cirrhosis suggests that other risk factors in addition to alcohol play a role in the pro g ression of the disease.
Studies in experimental animals other than primates have failed to pro d u c e either alcoholic hepatitis or cirrhosis, limiting their usefulness in studying disease pro g ression. Ne ve rtheless, animal studies have provided data on seve r a l i n t e r related mechanisms that may contribute to the development of alcoholic l i ver disease (Crabb 1993).

Alcohol Metabolism
The liver is the primary site of alcohol metabolism in the body. The main pathway of alcohol metabolism invo l ve s chemical reactions that are mediated by two enzymes (see figure 1 below ) . First, the enzyme alcohol dehyd ro g e n a s e (ADH) conve rts alcohol to acetaldeh yde, a highly re a c t i ve and toxic compound. Then, the enzyme aldehyd e d e h yd rogenase (ALDH) conve rt s a c e t a l d e h yde to acetate, which can be used as a fuel by the cell. During each of these steps, hyd rogen atoms are transf e r re d f rom the alcohol and acetaldeh yde (i.e., hyd rogen "d o n o r" molecules) to a hyd rogen "a c c e p t o r" molecule called nicotinamide adenine dinucleotide (NAD), which then becomes re d u c e d NAD (i.e., NADH). This chemical reaction is called an oxidation of alcohol and acetaldehyde. The extent of alcohol metabolism through this pathway is c o n t rolled by the ADH content of the l i ver and by the NADH/NAD ratio (also called the re d ox state) in the cell. The capacity of this pathway of alcohol metabolism is not influenced significantly by the level or duration of alcohol exposure.

Figure 1
The metabolism of alcohol by the alcohol dehydrogenase pathway. In the liver, alcohol is converted to acetaldehyde, and the acetaldehyde is converted into acetate. The enzyme alcohol dehydrogenase (ADH) assists the chemical reaction in (i.e., catalyses) the first half of alcohol metabolism, and the enzyme aldehyde dehydrogenase (ALDH) catalyzes the second half. Nicotinamide adenosine dinucleotide (NAD) is a co-enzyme that plays an accessory role in the reactions and accepts hydrogen atoms.
A secondary pathway of alcohol metabolism invo l ves an enzyme system c a l l e d the microsomal ethanol-ox i d i z i n g s y stem (MEOS), which helps eliminate many toxic compounds from the body and uses an enzyme called cytochro m e P450 (CYP 2 E 1 ). This enzyme, which c o n ve rts alcohol to acetaldehyde and uses nicotinamide adenine dinucleotide phosphate (NADP) as a hyd ro g e n a c c e p t o r, is induced by chronic alcohol e x p o s u re (Lieber and DeCarli 1970). Mo re ove r, CYP 2 E 1 is presumed to facilitate alcohol elimination, particularly at the high BACs that pre vail in heavy drinkers. Studies in rats found that in the presence of "f ree iro n" (i.e., iro n that is not bound to other molecules), C Y P 2 E 1 also generates various re a c t i ve oxygen radicals that can damage cellular components (Cederbaum 1987). C h ronic alcohol consumption can substantially increase iron levels in the b o d y. In fact, almost one-third of alcoholics have e xc e s s i ve iron levels in their livers, much of which is free iro n ( Nanji and Hi l l e r -Sturmhöfel 1997). Thus, these elevated iron levels may contribute to alcoholic liver damage.
Most acetaldehyde generated by ADH and CYP 2 E 1 is rapidly conve rted to acetate by ALDH. Some acetaldehyd e , h owe ve r, may combine with liver p roteins to form harmful compounds that can impair the function of va r i o u s c e l l ular components and enzymes. In addition, alcohol can combine with other molecules in the cell to form potentially dangerous compounds, such as fatty acid ethyl esters and phosphatidylethanol (see figure 2). As described in the section "A n i m a l Models of Alcoholic Card i o m yo p a t h y, " p p. 98-100, these compounds may damage the membrane that surro u n d s each cell, there by contributing to alcoholic heart disease. I n order to function, the body, including each individual cell, re q u i res large amounts of energy. This energy is supplied through the bre a k d own (i.e., metabolism) of nutrients, such as carbohydrates (e.g., sugars and s t a rch), proteins, and fats. Nu m e rous chemical pathways exist through which nutrients and other molecules (including alcohol) can be broken down. Many of these pathways include chemical reactions that invo l ve ox y g e n or hyd rogen atoms. These reactions are called ox i d a t i o n reactions. Generally speaking, oxidation reactions are those that add oxygen to or re m ove hyd rogen from a substance (or both). Thus, the metabolism of alcohol also invo l ves two oxidation reactions. First, the enzyme alcohol dehyd rogenase conve rts alcohol to acetaldehyd e by re m oving hyd rogen. Then, the enzyme aldehyd e d e h yd rogenase conve rts acetaldehyde to acetate by re m oving additional hyd rogen and adding ox y g e n .
Oxidation reactions, howe ve r, sometimes result not only in the formation of stable, nontoxic molecules but also generate highly unstable (i.e., re a c t i ve) molecules. Many of these molecules contain oxygen and are called oxygen radicals. Common oxygen radicals include superoxide (O 2 • ), hyd rogen peroxide (H 2 O 2 ), and hyd rox y l radicals ( • OH). If unchecked, these oxygen radicals can damage cells by attacking vital cell components, such as fat and protein constituents of the cell wall and the cell's genetic material.
Because the formation of oxygen radicals is a natural p rocess that occurs during many metabolic pro c e s s e s , cells have developed several pro t e c t i ve mechanisms to eliminate those radicals before they can do damage and to pre vent their formation. These mechanisms employ molecules called antioxidants, which can inhibit ox i d a -tion. Some antioxidants are enzymes found in the cells that steer the radicals into chemical reactions that generate n o n t oxic molecules. For example, the enzyme superox i d e dismutase helps conve rt superoxide radicals into water. Other antioxidants are compounds found in foods or generated by the body itself, such as vitamin E, vitamin C, and glutathione (GSH). These compounds have s e ve r al mechanisms of action. For example, GSH can n e u t r a l i ze oxygen radicals by transferring hyd rogen to the re a c t i ve molecules.
Using their internal antioxidants, cells can deal with normal levels of oxygen radical formation. When ox y g e n radical formation is greater than normal, or antiox i d a n t l e vels are lower than normal, howe ve r, ox i d a t i ve s t re s s occurs that may contribute to cell death (i.e., necro s i s ) a n d tissue damage, for example, in the live r. Chronic a l c o h o l consumption can increase ox i d a t i ve stress thro u g h s e ve r a l mechanisms. For example, animal models have demonstrated that alcohol metabolism is associated with the generation of oxygen radicals and that chronic alcohol consumption reduces the levels of antioxidant enzymes as well as of other antioxidants (e.g., GSH) (Colell et al. 1998; Nanji and Hi l l e r -Sturmhöfel 1997).

Oxidation and Formation of Free Radicals
What Is Moderate Drinking?

Generation of Oxygen-Derived Free Radicals
O x i d a t i ve stress may also play a role in the pathogenesis of alcoholic liver disease, and several mechanisms contribute to the generation of exc e s s i ve oxygen radicals or the reduction of antioxidant leve l s ( Ishi et al. 1997). The primary organelles that generate oxygen radicals during alcohol metabolism are microsomes and mitochondria. For example, rat live r m i c rosomes treated with alcohol generate a free radical, identified as 1-hyd rox ye t h y l radical, that can damage proteins as we l l as DNA. The alcohol-inducible enzyme C Y P 2 E 1 appears to play an import a n t role in the increased production of this radical, because radical formation was much higher in microsomes isolated f rom alcohol-fed rats (which showe d e l e vated CYP 2 E 1 activity) than in micros o m e s isolated from control rats (see Ishi et al. 1997 and re f e rences therein).
Studies on alcohol metabolism using perfused rat livers have noted an i n c reased production of a certain ox y g e n radical called superoxide anion (O2 • ) ( Bautista and Sp i t zer 1992). This radical is generated mainly in the pro c e s s that produces AT P, the cell's unive r s a l energy curre n c y, in the mitochondria. Acute alcohol administration can i n c rease superoxide generation in live r mitochondria (Sinaceur et al. 1985), and alcohol-fed animals develop stru cturally and biochemically abnormal mitochondria. These mitochondria h a ve decreased levels of proteins needed for ATP production and ox i d i ze va r ious compounds at reduced rates.
The mitochondria also contain the enzyme superoxide dismutase (SOD), which generates hyd rogen perox i d e radicals. No r m a l l y, this radical is degraded by a mitochondrial enzyme called glutathione peroxidase. In the presence of i ron, howe ve r, some of the hyd ro g e n p e roxide generates other highly re a c t i ve radicals that in turn can impair both the stru c t u re and function of the mitochondria. As mentioned above, iro n l e vels frequently are elevated in alcoholics. Fu rt h e r m o re, acute alcohol administration induces SOD activity and may, there f o re, promote tissue damage (see Ishi et al. 1997).
Another mechanism through which alcohol-induced ox i d a t i ve stress might result in liver disease invo l ves the enhanced metabolism of fat molecules (i.e., lipid peroxidation) (Muller and Siles 1987). Free radicals generated f rom ox i d a t i ve stress can re m ove electro n s f rom unsaturated fatty acids, re s u l t i n g in lipid radicals. The lipid radicals can react with oxygen to form lipid peroxide radicals, which, in turn, interact with other fatty acids, there by cre a t i n g a chain reaction of lipid perox i d a t i o n . Such chain reactions may generate biologically active compounds, such as molecules that cause widening or narrowi n g of blood vessels, there by incre a s i n g the risk of card i ovascular disease. Mo reove r, lipid peroxidation may generate molecules that re c ruit inflammatory cells (i.e., chemoattractant molecules) rendering the host more susceptible for i n f l a m m a t o ry attacks.
As discussed in the sidebar (see p. 96), the body has developed several pro t e c t i ve systems (i.e., antioxidants) that eliminate radicals, including glutathione (GSH) and vitamin E, as well as certain enzymes. When these pro t e c t i ve systems are ove rwhelmed, tissue dama g e f rom free radicals may result. Animal studies found that both acute and c h ronic alcohol exposure can interf e re with antioxidant activity. For example, large acute doses of alcohol re d u c e GSH levels in the liver by 25-50 percent (Crabb 1993), and GSH levels in the mitochondria of chronically alcoholfed animals are also reduced. Si m i l a r l y, long-term alcohol administration has been shown to decrease vitamin E

Figure 2
The presence of alcohol (i.e., ethanol) in tissues results in the generation of potentially toxic metabolites in the cells. Ethanol molecules combine with free fatty acids to form fatty acid ethyl esters, which have been linked to tissue injury. Ethanol also may combine with phosphatidylcholine (i.e., a membrane component) to form phosphatidylethanol, a compound that potentially alters cell membranes. Ethanol metabolism forms acetaldehyde and, potentially, free radicals, which may interact with cellular components and possibly interfere with their functions. l e vels in rat liver mitochondria. Thus, a reduction in antioxidant levels caused by chronic alcohol intake pre d i s p o s e s the liver (and possibly other organs) to attack by free radicals.

Endotoxins
Another mechanism that may contribute to alcohol-related liver damage invo l ve s bacterial endotoxins, which are molecules d e r i ved from the cell walls of cert a i n bacteria, including many that live in the intestine. When those bacteria die triggers the release of certain molecules (i.e., inflammatory cytokines) that serve to control the damage caused by endot oxin (e.g., tumor necrosis factor alpha [T N F -α] and prostaglandins). An ove r p roduction of TNF-α may damage hepatocytes. Both human alcoholics and experimental animals exposed to alcohol exhibit increased levels of endotox i n and inflammatory cytokines circ u l a t i n g in the blood (Nanji et al. 1993). This alcohol-induced increase in endotox i n l e vels may result from an enhanced " l e a k i n e s s" (i.e., permeability) of the intestinal wall and possibly from a reduced capacity to eliminate bacterial e n d o t oxin. Animal studies using intragastric alcohol administration have also demonstrated that Kupffer cells, p a rt i c u l a r l y with their production of p rostaglandin E (PGE 2 ) and TNF-α, contribute to endotoxin-mediated live r damage associated with alcohol intake (Thurman et al. 1998). One hypothesis suggests that the excess Kupffer celld e r i ved PGE 2 stimulates oxygen consumption in the hepatocytes in cert a i n a reas of the liver of alcohol-fed animals. This increased oxygen consumption may promote a temporary lack of ox y-gen (i.e., hypoxia) in the blood flow i n g t h rough those liver areas, followed by a return to normal oxygen levels in the live r. Such hypoxic episodes, in turn, might p romote the generation of free radicals.

Liver Regeneration
The live r's ability to regenerate itself is essential for allowing the organ to re c over from various forms of injury, including those related to alcohol. Fo r example, liver regeneration may help explain why only a re l a t i vely small prop o rtion of drinkers develop irre ve r s i b l e l i ver disease. By the same token, inhibition of liver regeneration by alcohol abuse may be an important factor in the p ro g ression of liver disease in humans. To study this issue, re s e a rchers surgically re m oved parts of the livers of alcohol-exposed and control rats. These studies found that both acute and chro n i c alcohol administration inhibit the production of new genetic material (i.e., DNA) in the regenerating liver (Yo s h i d a et al. 1997). Without sufficient DNA p roduction, howe ve r, the re m a i n i n g l i ver cells cannot multiply as rapidly to regenerate the live r. Other re s e a rc h e r s ( Zeldin et al. 1996) have inve s t i g a t e d the effects of acute and chronic alcohol administration on cytokine-inducible transcription factors. These transcription factors are re g u l a t o ry proteins that a re activated by cytokines and which are re q u i red for the conversion of genetic information into functional pro t e i n s during various processes, including live r regeneration. The investigators observe d that chronic alcohol administration impeded the ability of the cytokines to a c t i vate the transcription factors, there by limiting the generation of new liver cells.

Signal Transduction in the Liver
Alcohol intake can affect an organ indirectly by stimulating the release of hormones and other chemical messengers that influence a variety of cellular processes or by promoting exposure to harmful substances such as endotox i n s . In addition, alcohol can directly affect cells by interacting with and damaging components of the cell membrane ( p roteins and fat molecules [i.e., lipids]). This membrane also contains numerous docking molecules (i.e., re c e p t o r s ) that serve as point of contact for chemical messengers invo l ved in cellular communication, such as hormones. This interaction initiates mechanisms that translate chemical messages arriving at the cell membrane into cascades of biochemical reactions within the cell, e ventually resulting in changes in gene activity in the cell's DNA. This pro c e s s is known as signal transduction. Us i n g animal model systems, re s e a rchers have s h own that alcohol profoundly affects these communication processes (Ho e k et al. 1992). For example, acute alcohol e x p o s u re can activate several signal transduction systems, there by initiating a plethora of reactions within the cell. C o n ve r s e l y, prolonged alcohol exposure can cause the cells to become desensit i zed to the chemical messengers and impair cell activity.

Animal Models of Alcoholic Cardiomyopathy
Another organ affected by alcohol abuse is the heart. The typical form of alcoholic h e a rt disease, called dilated c a rd i o m yo p a t h y, is a degenerative disease of the h e a rt muscle (i.e., myo c a rdium). It is c h a r a c t e r i zed by a reduced capacity of the heart to pump blood (i.e., depre s s e d c a rdiac output), reduced ability of the h e a rt muscle to contract, widening (i.e., dilatation) of all heart chambers, and various other changes that occur on a cellular level (see figure 3). There are practical obstacles to studying the pathogenesis of this disorder in human patients, especially with obtaining tissue samples for such analyses. Some of these pro b l e m s can be re s o l ved using animal model systems (Richardson et al. 1998;Thomas et al. 1994), (For information on a chicken model of alcoholic card i o m yo p a t h y, see the article in this issue by Tabakoff and Hoffman, pp. 77-84). Re s e a rchers have not been able, howe ve r, to re p roduce true dilated card i o m yo p athy in animals. Fu rt h e r m o re, both in humans and in experimental animals, studies on alcohol's direct effects on the h e a rt in the intact organism a re complicated by alcohol's indirect effects on other organ systems and molecules that modulate heart function. For example, a l c ohol induces changes in the levels of catecholamines-an important class of chemical compounds, including dopamine, n o repinephrine, and epinephrine, that are i n vo l ved in normal cellular communication and which influence cardiac function.
A l c o h o l's direct effects on the heart can best be studied by studying perf u s e d h e a rts or isolated cardiac muscle pre p ar ations. In studies using perfused heart s , alcohol depressed a variety of indicators of the heart's ability to contract. Fo r example, the rate and extent to which p re s s u re developed in the heart chambers d e c reased and the rate at which the heart muscle subsequently re l a xed slowe d d own (Lochner et al. 1969;Segel 1988). Fu rt h e r m o re, in hearts perfused with alcohol, re s e a rchers observed an incre a s e d volume of blood in the heart's left ve n t r icle at the end of the heartbeat, when the chambers are re l a xed (i.e., the diastole) ( Segel 1988). These data suggest that alcohol interf e res directly with the contractile function of the heart muscle.

Mechanisms of Alcohol's Actions on the Myocardium
Animal studies have identified seve r a l mechanisms through which alcohol might affect the myo c a rdium. For examp l e , studies in rats demonstrated that acute alcohol intoxication can rapidly lead to a reduction in muscle pro t e i n p roduction (i.e., synthesis). Sp e c i f i c a l l y, acute alcohol exposure reduced the synthesis of myofibrillar protein, which is re q u i red for heart muscle contraction ( R i c h a rdson et al. 1998). Mo re ove r, c h ronic alcohol exposure led to a loss of m yofibrillar protein after 6 weeks. These findings are consistent with studies showi n g that myo f i b r i l l a ry protein loss occurre d in human alcoholics and that the remaining cardiac myo f i b r i l l a ry pro t e i n was in disarray (Hibbs et al. 1965).
Acute alcohol treatment also might reduce the heart's ability to contract by i n t e rfering with the process thro u g h which nerve signals induce the muscle to contract. For most muscles to contract, the brain emits a signal that is transmitt e d to nerve cells (i.e., neurons) that are in contact with the muscle cell. When receiving the signal, these neuro n s release a neurotransmitter (usually acetylcholine) that interacts with re c e ptors on the muscle-cell surface. This interaction causes a tiny electrical impulse at the muscle cell membrane that through a process called exc i t a t i o ncontraction (E-C) coupling, activa t e s p roteins within the cell that perf o r m the actual contraction (i.e., contractile p roteins). Calcium ions in the fluid filling the cell (i.e., the cytosol) play a key role in E-C coupling. When the cell "rests," calcium levels in the cytosol are l ow. The electrical impulse at the cell membrane, howe ve r, causes the re l e a s e of large amounts of calcium from storage compartments within the cell. This sudden increase in calcium levels allow s the contractile proteins to perform their function, leading to muscle contraction. To release the contraction and allow the muscle to relax again, the calcium is rapidly transported back into the storage compartments, resulting in a re t u r n of the calcium concentration to its normal, low levels. This temporary incre a s e in calcium levels followed by a rapid d e c rease is called a calcium transient.
Alcohol may interf e re with this E-C coupling by affecting the calcium transients (Thomas et al. 1994). Me t h o d s for measuring calcium in the cytosol of intact cells have allowed re s e a rchers to examine alcohol's effects on the calcium transients in isolated cardiac muscle cells (i.e., myo c y t e s ) and muscle fibers. Such studies have demonstrated that moderate to high levels of alcohol reduce the magnitude of the change in calcium levels in the cytosol in re s p o n s e to an electrical impulse (Thomas et al. 1994). In addition, other studies have s h own that muscle contraction i s i m p a i red by alcohol concentrations that do not affect calcium levels in the cytosol (Guarnieri and Lakatta 1990). These findings suggest that in addition to affecting calcium transients, alcohol reduces the sensitivity of the contractile p roteins to calcium.

Effects of Long-Term Alcohol Administration on Ca rdiac Fu n c t i o n
The effects of long-term alcohol consumption on cardiac function have been examined in a wide variety of ani-  mal models, including mice, rats, dogs, monkeys, and baboons. Most of those studies found that although metabolic changes occur within a few weeks of alcohol administration, signs of depre s s e d contractile function are detectable only after several months of alcohol consumption. Various studies found the f o l l owing effects of long-term alcohol c o n s u m p t i o n : • Rats receiving drinking water containing 30 percent alcohol water for 8 months exhibited an increase in the size of the left ventricle, indicating an enlargement of the heart. At the cellular level, prolonged alcohol administration led to a reduction in the total number of myocytes comprising the left ventricle (see Thomas et al. 1994 and re f e rences there i n ) .
• The left ventricle of dogs that re c e i ve d alcohol for 18 months displaye d i n c reased scarring of the heart tissue as evidenced by increases in total collagen levels, which may re d u c e the heart's ability to extend and contract during each heart beat (Thomas et al. 1980).
• Detailed studies of the stru c t u re of c a rdiac myocytes in alcohol-fed a n imals found various stru c t u r a l c h a n g e s that are similar to some defects found in human patients with alcoholic card i o m yo p a t h y.

Effects of Alcohol Metabolism on the Heart
Animal model systems have also helped elucidate the metabolic effects of longterm alcohol exposure on cardiac function (Thomas et al. 1994). For example, comp a re d with normal heart muscle, alcoholic heart muscle may have a re d u c e d capacity for ox i d a t i ve metabolismthat is, the process that generates energy by breaking down the sugar glucose. A l t e r n a t i ve l y, alcoholic heart muscle may generate additional energy in the absence of oxygen (i.e., nonox i d a t i ve l y ) by breaking down glycogen (i.e., a storage form of glucose in the cell) to lactic acid. Such a change in metabolism may explain the more acidic intracellular e n v i ronment observed when the workload is increased in perfused hearts of alcohol-fed hamsters compared with c o n t rol animals. Fu rt h e r m o re, alcohol metabolites also may adversely affect c a rdiac function, as follow s : • Ac e t a l d e h yde may affect card i a c function indirectly by enhancing the release of catecholamines.
• Fatty acid ethyl esters, which accumulate in the hearts of alcohol-fed animals, may damage mitochondria and alter the pro p e rties of biological membranes (e.g., membranes s u r rounding the cells or va r i o u s organelles) (Laposata 1998), there by impairing cell function.

Contractile Function
Long-term alcohol consumption also i n t e rf e res with the heart's contractile function (Thomas et al. 1994). Fo r example, in dogs, long-term alcohol feeding lowe red the rate with which p re s s u re developed in the left ve n t r i c l e and increased pre s s u re in the left ve nt r icle at the end of the diastole. Re s e a rc h e r s made similar observations in rats. Mo reove r, dogs that we re fed alcohol for 1 year developed inefficient, random contractions (i.e., fibrillation) in the ventricle more easily than did contro l animals that re c e i ved no alcohol. As in humans, acute alcohol administration to animals that had previously consumed alcohol chronically further incre a s e d the animals' vulnerability to fibrillation. He a rt function has also been examined in isolated heart muscles derive d f rom alcohol-fed animals. In isolated muscles from the left ventricle of rats that had been fed alcohol for 5 weeks to s e veral months, one or more indicators of the heart's mechanical perf o r m a n c e we re significantly reduced. In studies using isolated perfused hearts, howe ve r, a species difference existed with re g a rd to the effects of long-term alcohol feed-ing. Thus, w h e reas perfused heart s f rom hamsters exhibited a reduction in p re s s u re during contraction of the h e a rt (i.e., systolic pre s s u re) and an i n c rease in pre s s u re during the re l a xation of the heart (i.e., end-diastolic p re s s u re), hearts from rats showed little change in contractile pro p e rties after c h ronic alcohol administration.
In summary, scientists can pro d u c e alterations in heart function in animal models that are similar to those found in human alcoholics. Animal models do have some limitations, howe ve r. Fo r example, re s e a rchers have not been able to re p roduce alcoholic card i o m yo p a t h y with its reduced output. Mo re ove r, in contrast with alcoholic card i o m yo p a t h y in humans, all cardiac effects observe d in animal models can be re versed by withdrawing the alcohol.

Animal Studies of FAS
In humans, alcohol exposure during gestation has been associated with a variety of negative outcomes, including c o g n i t i ve and behavioral deficits, comp romised growth, and death during late pregnancy or soon after birth (i.e., in the perinatal period) (St reissguth et al. 1980). The most seve re manifestation of prenatal alcohol exposure is FAS, which is characterized by a re c o gnizable cluster of facial malformations (i.e., facial dysmorphology), grow t h re t a rdation, and mental re t a rd a t i o n . Animal studies on FAS have adva n c e d re s e a rc h e r s' knowledge in these areas, as well as of the effects of prenatal alcohol e x p o s u re on the immune system, hormonal (i.e., endocrine) systems, and central nervous system. Most of these studies have been conducted in mice and rats that re c e i ved alcohol, although re s e a rchers have used other species as well (Guerri 1998;Hannigan 1996).
Some problems exist when using animal models to investigate alcohol's effects on the developing fetus. For example, the gestational process differs somew h a t among different species. Thus, the major g rowth spurt in the human fetal brain occurs during the third pre g n a n c y t r i m e s t e r, whereas the corre s p o n d i n g g rowth spurt in mice and rats occurs after birth. Re s e a rchers must consider such differences when designing experiments and alcohol administration schedules. Ne ve rtheless, as described in the foll owing sections, alcohol's effects on the fetus are qualitatively similar in humans and experimental animals.

Prenatal and Postnatal Growth Retardation
Babies born to alcohol-abusing women f requently have a lower than normal birt h weight. Theore t i c a l l y, this growth re t a rd ation could result from malnutrition of the mother rather than from prenatal alcohol e x p o s u re itself, because alcohol-abusing women are less likely to eat a nutritious diet during pre g n a n c y. Re s e a rchers have i n vestigated this issue in rats and mice, using various modes of alcohol administration. The resulting findings suggest that fetal alcohol exposure per se rather than maternal malnutrition causes reduced birth weight (see No rton and Kotkoshie 1991 and re f e rences therein).

Facial Dysmorphology
The distinctive facial features associated with FAS include small eye openings, a small nose, a flattened gro ove betwe e n the upper lip and the nose (i.e., philtru m ) , and an abnormally small upper jaw (i.e., maxillary hypoplasia). To determine whether these features could be re p roduced in an animal model, re s e a rc h e r s a d m i n i s t e red alcohol by a stomach tube to pregnant monkeys once a we e k during gestation. The offspring of these animals exhibited several of these FA S characteristics (i.e., a small nose, poorly defined philtrum, and maxillary hypoplas i a ) ( C l a r ren and Bowden 1982). Si m i l a r o b s e rvations we re made in mouse e m b ryos exposed to alcohol at a specific time during gestation (Sulik et al. 1981). These findings confirm the o b s e rvations in humans that alcohol e x p o s u re during pregnancy can pro d u c e the characteristic facial dysmorphology in the developing fetus.
Re s e a rchers also have begun to identify the mechanisms underlying alcohol's effects on facial characteristics, such as alcohol-induced cell death. Studies in mouse and chick embryos re vealed that this cell death primarily occurs in specific g roups of cells, particularly in a gro u p called neural crest cells (Smith 1997). A subset of these cells forms va r i o u s facial stru c t u res, such as the jaws, nose, ears, orbital bone around the eye, and f o rehead. When chick embryos we re exposed to alcohol during a specific (and ve ry short) time period early during embryo development, cell death o c c u r red among the neural crest cells, resulting in cell loss and, consequently, facial malformation (Smith 1997). In the neural crest cells, alcohol may exe rt its deleterious effects by interfering with the formation of a substance called retinoic acid, a vitamin A deriva t i ve that is necessary for the development of those cells into facial feature s ( National Institute on Alcohol Ab u s e and Alcoholism [NIAAA] 2000).
of vesicles in the remaining synapses. These changes may indicate that the c o rtical synapses have re m a i n e d i m m a t u re, which may result in learning disabilities.

Hi p p o c a m p u s .
The hippocampus is a brain area associated with learning and m e m o ry. One of the major functions of the hippocampus is to constru c t "spacial cognitive maps" of the surroundings. Ma t h ews and Mo r row (2000) recently re v i ewed the effects of acute and chronic alcohol exposure on the hippocampal function. In rats, gestational alcohol exposure reduced the number of certain neurons in the hippocampus by 20 percent. This re d u c t i o n may contribute to some of the cognitive deficits observed in alcohol-exposed infants (Barnes and Walker 1981).
Ce re b e l l u m . The cerebellum is an are a located at the base of the brain that is i n vo l ved in controlling posture, balance, and coordination. Whereas in humans the cerebellum deve l o p s largely during the prenatal period, neuron production and development in rats continue into the postnatal period. Consistent with this deve l o p m e n t a l pattern, alcohol administration to rats during gestation did not alter the number of specialized cells in the cere b e llum (i.e., Pu rkinje cells), although some s t ructural changes occurred in t h o s e cells, and the maturation of cert a i n c e l l components was delayed compare d with control rats. These differences, howe ve r, d i s a p p e a red by day 17 after birt h . C o n ve r s e l y, alcohol exposure thro u g h o u t gestation and into the early postnatal period reduced the number of synaptic contacts in a particular subregion of c e rebellum (i.e., the molecular laye r ) and the number of Pu rkinje cells (No rt o n and Kotkoskie 1991). These changes may contribute to the impaired motor c o n t rol (e.g., balance deficits) observe d in alcohol-exposed children.

Neurochemical Alterations
Animal studies have shown that gestational alcohol exposure not only affects n e u ronal development but also neuro n a l function, as indicated by alterations in n e u rotransmitter levels. One import a n t n e u rotransmitter is acetylcholine, which a l l ows communication among neuro n s as well as between neurons and muscle cells. Ac e t y l c h o l i n e's actions are re g u l a t e d by an enzyme called acetylcholinesterase. When rat fetuses are exposed to alcohol during the final week of gestation, postnatal levels of acetylcholinesterase are reduced throughout the brain (Ru d e e n and Guerri 1985). These results suggest the possibility that prenatal alcohol e x p o s u re delays the development of or permanently alters acetylcholine-using n e u ronal systems in the brain.
Animal studies also found that prenatal alcohol exposure reduces the leve l s of the other neurotransmitters. Fo r example, in rats, ve ry early prenatal alcohol exposure significantly delays the d e velopment of serotonin-using neuro n s in many brain regions during gestational periods that are most likely critical for normal brain development (NIAAA 2000). Si m i l a r l y, prenatal exposure to e ven moderate alcohol levels causes d e c reases in the number and function of c e rtain receptors for the neuro t r a n s m i tter glutamate (NIAAA 2000). If these effects occur during critical periods of brain development, they may contribute to the mental and behavioral deficiencies associated with FAS. Mo re ove r, both prenatal and postnatal alcohol e x p o s u re alters the levels of the neurotransmitter gamma-amino butyric acid (GABA), although the direction of these changes varies among different brain regions. Thus, GABA levels increased in some regions (e.g., the frontal cort e x and the amygdala), decreased in some regions (e.g., the hippocampus), and remained unchanged in other re g i o n s (e.g., the hypothalamus and septal are a ) (Ledig et al. 1988). In general, alcoholinduced reductions in neuro t r a n s m i t t e r l e vels are most pronounced when alcohol exposure occurs throughout gestation and into the postnatal period ( No rton and Kotkoskie 1991).
In addition to alcohol's direct tox i c effects on brain development, indire c t effects, such as alcohol-related nutritional deficiencies (e.g., zinc deficiency) may contribute to alcohol's adverse effects on fetal development (No rton and Ko t k o s k i e 1991). Fu rt h e r m o re, re s e a rchers have postulated that alcohol's toxic effects on the placenta and alcohol-induced fetal h y p oxia play a role in the pathogenesis of central nervous system malformations associated with FAS.
In summary, animal experiments h a ve clearly demonstrated that the fetus is susceptible to various types of damage during gestational alcohol exposure and that the extent of damage is related to the dose and extent of alcohol exposure .

Conclusions
Nu m e rous animal model systems have contributed substantially to an understanding of the pathways by which alcoh o l might cause organ damage, such as alcoholic liver disease and card i o m yo p a t h y, as well as FAS. Ne ve rtheless, the ability of these models to mirror these conditions in humans is somewhat limited. For example, whereas many alcohol-induced conditions in humans a re irre versible (e.g., cirrhosis and card i o m yopathy), re s e a rchers have been unable to produce such irre ve r s i b l e organ damage in animal models. On e reason for this inability may be that such a l c o h o l -related disorders in humans take many years of alcohol consumption to develop in humans. In addition, diff e rences among species may exist in disease pathogenesis.
Another limitation of animal models is the difficulty in duplicating the circ u ms t a n c e s under which alcohol consumption occurs in human alcoholics. Mo s t experimental systems are designed to assess the effects of either acute or chro n i c alcohol exposure. Acute models analyze the immediate effects of alcohol or its metabolites on cellular function or molecular organization. Such experiment a l situations may indeed re p roduce the effects of acute alcohol intake on human organs and tissues. Conve r s e l y, chro n i c models are concerned with the longterm adaptive or injurious responses of organs and cells to persistent alcohol e x p o s u re. Long-term alcohol consumption in humans can va ry substantially over time with respect to the amount and frequency of alcohol intake. These variations are difficult to mimic in animals. Fu rt h e r m o re, many of the changes produced by chronic alcohol e x p o s u re (e.g., cirrhosis and card i o m yopathy) are re versible in experimental animals, whereas in humans these conditions are permanent. Ac c o rd i n g l y, re s e a rchers cannot be absolutely cert a i n of the veracity of their models. In addition, it will be difficult to determine whether chronic diseases associated with alcohol abuse in humans re f l e c t the accumulation of numerous acute injuries or whether they are independent of acute effects and caused by as ye t u n k n own factors. Ne ve rtheless, a better understanding of the alcohol-re l a t e d maladies clearly depends upon re s e a rc h e r s' ability to learn more about the molecular and cellular events associated with alcohol consumption. Some of these e vents reflect direct interactions betwe e n alcohol and various cell constituents (e.g., proteins, fats, and DNA), where a s other effects are indirect, acting thro u g h multiple biochemical cascades. All of these interactions are similar in humans and laboratory animals. Thus, although a need remains for improved animal models that mimic the human c o n d itions more closely, existing models a re capable of providing considerable information re g a rding the mechanisms by which alcohol injures the body. s